Diabetes is an immense healthcare burden, with great costs for management of this disease and its associated complications. Human pluripotent stem cells (PSC)s, including embryonic stem (ES) cells and induced pluripotent stem (iPS) cells offer great potential for the study and treatment of human disease. Rodent models have also been critical in understanding pancreas development and generating models of diabetes. Here, we propose to use both complementary systems to study human pancreatic development and function, with a focus on the beta cell, which regulates blood glucose via insulin secretion. While rodent studies have produced tremendous advancements, human and mice are not the same and it is important to define differences in pancreas biology as these could impact the treatment of diabetes. For example, heterozygous mutations in the transcription factors GATA6 or GATA4 have been shown to cause pancreas agenesis in humans, with mutations in GATA6 being the most common cause of this genetic disease (~50%). In contrast to the human phenotype, mice heterozygous for either GATA6 or GATA4 have no pancreatic phenotype. Compound mutations of GATA4 and GATA6 do mimic the human disease suggesting an overall conservation in GATA family members in pancreas development but with humans being much more sensitive to dosage of these genes. Retinoic Acid (RA) signaling is a critical mediator of pancreas development both in rodent models and is a required signaling input in directed differentiation protocols generating beta cells from human PSCs. RA is also a known regulator of GATA6 and GATA4 expression. We will examine the interplay between RA and GATA4/6 in both the mouse and human PSC model systems. Specifically, will use mutants in GATA family members as well as dominant negative RXR constructs in both systems, assessing conservation and differences in their impact on pancreas specification and endocrine cell development. Lastly, we will study a single nucleotide polymorphism in the 5? GATA6 enhancer, which in preliminary data we demonstrate regulates GATA6 expression and pancreas development in the human PSC system. These studies will help define the developmental and functional differences between human and mice beta cells that have the potential to directly impact the treatment of not only rare genetic forms of diabetes but also reveal unique human beta cell biology with implications in the management of both type 1 and type 2 diabetes.